Characters of expression
vector depend upon the kind of biosystems one uses and what you want from such
structure. For prokaryote one requires one kind of vectors and for eukaryotes
one may require different vectors depending upon whether it is a mammalian
cell, insect cell line or plant tissues.

·Whatever
may be the system, the basic requirement for any expression system is a
promoter cloning site(s) next to it and transcriptional terminator. The promoter
is the region, which is recognized by the RNA polymerase and initiates
transcription and transcribes any on its way. As the transcription progresses
it has to have region where the transcription has to be terminated, if the
cloned material has such sequences it is OK other wise one has to provide such
sequences for transcriptional termination.

·In addition
the vector should have an origin suitable for replication initiation in a
particular bacteria or any host and one desires to have an origin which produces
high copy numbers. If the vector requires an origin for another system one has
to provide suitable origin of eukaryotes also. Then such vectors become
shuttle vectors.

·The vector
has to have a selection marker gene such as antibiotic resistance gene suitable
for the host.

A
gene with its promoter, operator, coding and terminator regions- structural
features of a Gene

·Another
most desirable feature of the vector is to have regulator elements so that one
makes the gene to express when one needs.

·In addition
one may have to use certain sequences between the promoter elements and the
insert, which on transcription provide sequences for translation that may help
targeting the protein to certain destination.

·The most
important aspect of cloning into a vector is to align the insert DNA in such a
way that once the promoter is used for initiating transcription, the transcript
should possess a proper leader sequence containing Shine Delgarno for bacteria
and Kozak sequence for eukaryotic systems for initiation of translation. The
ultimate aim for cloning is get an expressed product from the cloned DNA
material.

·One has to
choose a promoter for expression for a particular tissue. It should have
surrounding elements which respond to certain stimulus or may act as enhancer
sequences which help is the efficiency transcription.

I. Prokaryotic
Expression Vectors:

pLac-Z expression
vectors:

·Lac –Z
promoter operator is in frame with lac-Z alpha fragment (the NH3 terminal part
of Galactosidase gene. Multiple cloning sites are found in the border of NH3
end including ATG sequence. The presence of such restriction site sequences should
not disturb the functional activity of the protein, which complements with the
omega fragment of the Lac-Z produced by the bacterial cell as the complement.
If any gene is placed in proper frame in the MCS the protein expressed will be
is fused form. The expression of the gene can be regulated by IPTG (Isopropyl
thio b-Galactoside).

pET Expression
Vector:

·The size of
the vector is 5700bp

·It has T7
promoter adjacent to lac operator.

·Next to it
is a sequence called Shine Delgarno sequence.

·Adjacent to
S/D sequence there are few cloning sites such as Nde I, Nhe I and BamH I, at
the end of which is T7 phage transcriptional terminator is found.

·For the
expression of this gene the bacterial cell should provide T7 RNA polymerase,
which is under the control of Lac-Z operator/promoter.

· The
repressor produced by the bacterial lac-IQ can be regulated by IPTG.

---T7
P—Lac-O—S/D—(sequence tags)-Nde I-Nhe I-BamH I----T/t--,

(T/T= Transcriptional
terminator)

---T7
P—Lac-O—S/D—(sequence tags)-Nde I-Xho I-BamH I----T/t-,

The
pET Vector. This plasmid contains a drug resistant marker for ampicillin
resistance (green), thelacIgene (blue), the T7 transcription
promoter (red), the lac operator region (pale green) 3' to the T7 promoter, and
a polylinker region (black). Also, there are two origins of replication - one
is the f1 origin which enables the production of a single stranded vector under
appropriate conditions, and the other is the conventional origin of
replication. This image was used with permission from Dr.
Michael Blaber, of Florida State University. pET
expression Vector: http://www.bio.davidson.edu/

pTac expression Vector:

·This has
the combination of Trp and Lac-Z promoter –operator elements, which is
inducible. It is considered to be a very efficient promoter.

·Maltose
binding protein sequence is added next to the promoter for in expression mode.

·A lac-Z
alpha part is added to the maltose binding protein as a fusion protein.
Maltose binding protein is 42 KD.

·A multiple
cloning site is inserted in the N- terminal part of the lac-z alpha segment, so
any gene introduced in this site produces white colonies, at the same time it
generates fusion protein.

·Such
proteins can be purified on maltose columns for the columns are loaded with
certain factors, which can cleave the protein at required position.

·Clones can
be identified by immunoprecipitation against Matose binding protein or His tag

General notes:

A gene or open reading frame is
inserted into a restriction site of the vector polylinker, in the same
translational reading frame as the malE gene (encoding maltose-binding
protein). Insertion of the DNA fragment interrupts the malE-lacZa
fusion pre-existing on the vector, affording a screen for inserts on the proper
indicator plates. The fusion protein thus produced can be purified by amylose
affinity chromatography. The sequence coding for the four amino acids Ile-Glu-Gly-Arg is present just upstream of
the XmnI site. This allows the protein of interest to be cleaved from
maltose-binding protein with the specific protease Factor Xa. Fragments
inserted in the XmnI site (cleaves GAAGG↓ATTTC)
will produce a fusion protein that, after Factor Xa cleavage, contains no
vector-derived residues on the protein of interest.

Purified Maltose binding protein

Fusion protein
products: Histidine Tag:

·It is
possible to introduce certain sequences in frame so the cloned gene product is
produced as fusion proteins. The N-terminal part of the expressed protein is
used for purification. Such tags can be Histidine tags the fused protein can
be purified using Nickel column. A sequence such as NH3-L-V-P-R-G-S
* (MCE), the fusion protein is cleaved with Thrombin which cuts the
protein at serine (*). Similarly one can introduce sequences such as
NH3-I-E-G-R*-(MCE), which can be cut with Factor Xa, the position of cutting is
marked by (R*). Introduce desired DNA in th reading frame with in the cloning
sites

·Inserting
the coding segment of the DNA into these will generate a proper

One can generate fusion
products to be purified by adding sequences before the actual start codon of
the desired gene. The product can be targeted to be secreted into periplasmic
space or to be targeted into cell inclusions or the protein can be purified by
column chromatography or by cutting the tags by using certain specific
proteases or chemicals.

·While
cloning one can use expression cassettes for the desired purpose.

This unit
describes how pGEX vectors can be used in bacterial systems to express
foreign polypeptides as fusions with glutathione-S-transferase
(GST). In general, such fusion proteins are soluble and are easily purified
from lysed cells under non-denaturing conditions by absorption with
glutathione-agarose beads, followed by elution in the presence of free
glutathione. Potential applications of the pGEX vectors include the
expression and purification of individual polypeptides (including short
peptides) for use as immunogens and as biochemical and biological
reagents, and in the construction of cDNA expression libraries. This
protocol describes production and screening of pGEX transformants and
purification of milligram quantities of fusion proteins from 1-liter
cultures. The commentary describes several modifications to the expression
and purification protocol that may be useful in cases where fusion
proteins are insoluble or unstable.

Some of the sequences that generate
fusion proteins can be cut with specific reagents or enzymes to release the
gene product.

------D*P----, cut with acid (acid pH,
70%formic acid 48hrs).

------N*G----, cut with hydroxylamine pH 9,
at 45^oc 4hrs.

------R*--- or
---K*----, cut
with Trypsin pH 7-9.

------R*-----, cut with Clostripain pH8.

------N-N-N-N-K*, ---cut with Enterokinase.

------G-P*---, cut with collogenase.

------F-A-H-Y*--, - cut with H64A Subtisilin pH
8.

------L-V-P-R-*-G-S, cut with thrombin.

------R*-X—, endo Proteinase.

------G*-X, cut with endopeptidase (V8
protease).

------E-*A—, can be cut with STE
diaminopeptidase.

------Y*-K*-R—, KEX 1 protéase.

Secretary signals
to bacterial periplasmic space:

M-K-Q-S-T-I-A-L-A-L-L-P-L-L-F-T-P-V-T-K-A-*-R—

II. Eukaryotic
expression vectors:

The design of
eukaryotic vectors depends upon the type of system or tissue used and what gene
to be expressed and in what way.

In most of the
cases the eukaryotic expression vectors designed for manipulation in the
bacteria to begin with, it is only then they are transferred to eukaryotes. So
the vectors are called shuttle vectors.

Somme of the
vectors are designed to be integrated into the host chromosomal DNA and some
are designed for transient episomal expression.

·The
desirable size of the vector should be as small as possible.

·It
should contain replicative origin for bacterial system as well as eukaryotic
system one chooses.

·Vector
should contain a strong promoter, which can be a general promoter to be
expressed in all types of cells or the promoter should be tissue specific type
to be expressed only in kind of tissue.

·Another
desirable feature is in having regulatory or regulatable promoter (like
inducible or repressible), such as having response elements, activator
sequences or enhancer sequences or the combination of them.

·For
screening and selecting transformed cells a desirable marker gene inclusion in
the vectors is almost essential.

·Cloning
site or cloning sites next to the promoter elements has to be designed in such
a way it should generate a proper leader sequence at the beginning of the
transcript perhaps with Kozak elements and there should be a transcriptional
terminator sequence such as pol (A) signal block.

·Depending
upon the needs one can introduces sequences for targeting the gene product to
specific destinations.

·In
another novel type of situation it is possible to create an artificial
chromosome similar to that of YAC, where desired genes are introduced under the
control of regulatory elements for expression.

III.Yeast expression vectors:

·Yeast is a
Eukaryotic system.

·It is a
unicellular organism.

·One can
obtain mutants such as nutrient mutants or and conditional mutants.

·Mutant can
be maintained.

·They can be
grown into large numbers and density.

·Like other
eukaryotic systems the proteins fold properly.

·The system
provides glycosylation of proteins, but it may not be similar to mammalian
system.

·The
introduced gene product may be made to secrete it products into medium.

·Transformation
and culturing of the cells is relatively easy and cost effective.

Yeast Integrative plasmids (YIP):

·The size of
the vector is 5.5kbp,

·It has an
Ori site for replication and a selection marker gene (Ampicillin gene) for
E.coli cells.

·Plasmids
are unable to multiply in yeast cells.

·The plasmid
has URA3 selection marker gene for integration into the host nuclear DNA by
homologous recombination. A defective gene of the host is replaced by
functional gene. Similarly one can knock out a functional gene.

·One can
insert a gene in expression mode with border sequence which are homologous to
certain hast DNA sequences. There are few Restriction sites such as Puv II,
E.coli I, Cla I and HinD3 into which such expression cassettes with homologous
border sequences and a desired gene can be introduced.

===Homologous
DNA I--P------X gene----I-homologous
DNA===

·The border
sequences help in integration of the cloned gene into host chromosomal DNA.

·If the
border sequence of a host gene and new gene is introduced in between them, by
homologous recombination a host cell gene removed and in its place a foreign
gene gets inserted. This leads to gene knock out

.

Such
gene knockouts will enable researchers to understand the effect of single gene
on an organism.

~~---Ori—Amp^+--R1—Cla
I—D3---URA3—Sma I/Xma I--~~

~~
= Ends of the plasmid which is in circular mode.

URA3
acts as the homologous segment.

·Once the
gene is integrated by amplifying the clonal cells, one can go on and on in
producing the required product.

Yeast Episomal
expression vector:

·The plasmid
is circular mode.

·The plasmid
size is 7.769 kbp.

·It has ColE
1 origin for bacterial manipulation.

·It has
bacterial selection marker gene.

·It has URA3
as a selection marker gene for yeast cell. It has 2u plasmid DNA segment with
STB chunk (2000bp long) for replication in yeast cells. This helps for the
plasmid to replicate in the nucleus and produce a copy number of 100-200 per
cell.

·This high
copy number will produce greater yield of the gene product.

·It also
contain few cloning sites such as Sma I / Xma I, Bpu I, Cla I and Aat II.

·Into these
sites one can clone a gene in expression mode using a expression cassette.

·Yeast autonomously
replicating plasmids’ ARS sequence component is present, while in yeast
episomal plasmid a large component of 2u DNA is present. But adding CEN
sequences it can stabilize YRS plasmid, which is called “YCP”.

Using
such plasmid with expression mode Zn / Cu Super Oxide Dismutase (SOD) gene has
been cloned under the promoter of PGALD. This expression vector in yeast
produced a genuine protein used on patients with cross-reactions. Such
plasmids have been used in the production Hirudin an anticoagulant produced by
leaches called Hirudo medicinalis. When Hirudin is used as anticoagulants they
did not elicit any immune responses. Both these products are used during
operation.

During
blood transfusion ionic radicals are produced. They are very dangerous for
they react with other cellular enzymes and compounds. But Super Oxide
Dismutase reacts with super oxides and converts them into H2O2,
which is then cleaved into H2O and O2 by another enzyme
called Catalase. The SOD can also be used against inflammation disease such as
Osteoarthritis, Rheumatoid arthritis, Scleroderma and similar disease.

Though
the yield of required proteins is good, in certain cases the glycosylation is
not same as that of human products; so one has to resort to cell lines, which
produce products similar to human proteins.

Some
of the yeast expression vectors used contains specific promoters, which are very
efficient, and they can also be regulated.

For
example an expression vector for integration was developed for another yeast
related fungus called Pichia pastoris. This vector has a promoter derived from
Alcohol oxidase or ethanol oxidase, which is inducible. Next to the promoter
it has cloning site and transcription terminator sequences. The vector also
has Aox 5’ and Aox 3’ sequences for integration and a functional gene His for
selection. For transformation of Pichia pastoris- yeast cells, the vector is linearized.
These cells are highly productive and respond very well for induction with
alcohol. Using such systems Hepatitis B surface antigens were produced on
large scale.

A list of yeast
promoters:

The basic construction of the
vector is-

---Promoter-----X-gene—Ttr--

Promoter

Full name

Fold of induction

PGK

Phospho Glycerate kinase

20 fold

ADH1

Alcohol dehydrogenase, induced by ethanol and glycerol,
suppressed by glucose

20 fold

GAP

Glyceraldehyde 3-P dehydrogenase

20 fold

GAL1

Galactose utilizing

1000 fold

GAL10

Galactose utilizing

ADH2

Alcohol dehydrogenase, repressed by glucose

100

PHO5

Acid Phosphotase

200

MFalpha1

Mating factor a 1

105 fold if the TM is

shifted to24oC

AOX1

Alcohol oxidase

ADH/GRE

ADH growth hormone response element

ADH/ERE

ADH-estrogen hormone response element

PGK/ alpha2

100fold by shifting the temperature

GAP/GAL

150-200 induced by Galactose

CYC1/GRE

50-100 by deoxycorticosterols

PGK: I-----P-----I-mcsI---//--T/t.

ADH1: I------P-----I-mcsI--//---T/t.

GAP: I------P-----I-mcsI---//T/t.

GAL1: IUASI--I---p---I-mcsI---//T/t.

AOX1:I------P—I-mcsI----//-T/t (Pitchia pastoris)

Intracellular expression:

Eliminate
all UTR sequences upstream of ATG.

Precede
initiator codon with AAAAAATG.

The
gene cloned should be free from introns.

Transcriptional
terminator region should be GC rich.

For secretion purpose:

Use
yeast signal peptide sequences at the 5’ end of ATG.

MFalpha2-
by kex2 protease- lys-Arg*--

STE
diamino peptidase--*Glu-*-Ala-

Kex1-
-*Lys-*Arg-

6. Vectors for animal cells:

Human Papova viral based Vectors:

·They are
often called BKV vectors. BKV viruses are icosahedral, 50nm thick and ~5000bp
long.

·The viral
vectors can be maintained in human cell cultures as extra chromosomal elements
with high copy numbers.

·The vectors
developed are shuttle vectors.

·The vector
contains a large fragment of viral origin and some segment of DNA, which
provides elements for high copy numbers.

~~---Dre-MMtr-P—gene-X-Ttr—SV40-P—Kan+ gene- Ttr—BKV-Ori--~~

·Circular
module.

·A promoter
from Mouse mammary tumor virus has been included.

·The
promoter is abutted by a dioxin response element (DRE). Dioxin is orange-G
(2,3,7,8 Tetra Chloro di Benzo p-Dioxin (TCDD), it is a toxic substance used by
USA army in Vietnam during 1970-73.

·Next to it
is cloning site(s) and a transcriptional terminator signal sequence.

·Kanamycin
gene resistant gene is placed under Sv40 early promoter.

·It also
contains Ori-E and Ampicillin resistant gene.

·When this
vector containing a gene is transferred to a human cells, the vector enters
into the nucleus and copies into high copy numbers, the number can as high as
2000-8800 per cell.

·As the
number increases to the maximum the expression of the gene is induced by adding
Dioxin.

·The target
for the protein can be designed by using specific signal sequences before the
inserted gene in reading frame.

BKV derived composite Shuttle Vector for immunoglobulin subunits:

This
kind of vector was used for the production of light chain and heavy chain of a
specific antibody. The IgG produced were non-reactive when delivered into
human system.

1 st vector (pL):

It has
ColE-1 origin.

It has
AMP+ gene.

It has
promoter derived from b-Actin.

It has
T/tr region.

The Light
chain gene has been inserted in proper reading frame.

A gene
for DHFR has been put in place with a promoter derived fromSV40.

It
also contained BKV origin with high copy number element.

2nd Vector (pH):

It
contained ColE 1 origin and an Amp + gene.

It has
promoter from b-Actin gene where the IgG’s
heavy chain has been cloned with T/tr region at its end.

Kanamycin
plus gene has been placed under MM tr promoter.

Along
with BKV origin and high copy number element has been incorporated.

When
both these constructions are transfected and selected on Methotrexate and
Kanamycin. In fact in the presence of Methotrexate the DHFR gene by unknown
mechanism amplifies to high numbers.

Using
this technique researchers raised IgGs against the receptors found on malignant
lymphoma cells, also called Hodgkin lymphoma. However the IgG genes were so
modified, when they were introduced into human body without any immune response
against IgG, so they are called “Humanized antibodies”.
In this case IgGs raised are against malignant lymphomas cellular surface
glycoprotein. The surface antigens actually facilitate and activate cell for
further proliferation by cell-to-cell contact. Blocking such lymphomas by IgGs
prevents the further proliferation and surprisingly the malignant cells
regressed.

Similarly
one can use Glutamine synthase as selection marker gene, which is resistant to
Methionine sulfoximine (MSX). In this case the cell need lack this gene.

SV40 derived vectors:

SV 40
has late promoters and early gene promoters; they can be used for generating a
functional vector, which can be propagated in monkey or human cell lines. The
virus is isometric and it non-enveloped virus, It is made up of three types of
capsid proteins and all are derived from the same transcript by alternate
splicing. The DNA is double stranded, and circular, 5243 bp long. The virus
infects through cellular receptors, and the viruses are taken in by
endocytosis. Inside the cytoplasm the DNA is delivered into the nucleus. As
the ds viral DNA enters the nucleus it gets associated with host histones and
becomes a mini-chromosome. The DNA has an approximately 250-300 bp long origin
abutted on either side by capsid protein gene on the left side and T-antigen
gene on the right side of it. The genes slightly overlap the origin. The
or5igin consist of two 72 bp long enhancer regions, next to it is six copies GC
rich sequence and then it has 5 pentameric sequence and then it has AT rich
imperfect palindrome sequence. The TATA region id found with in the pentameric
sequences. This region acts as early promoter for the expression of T-Antigen
gene, which on expression produces T-antigen, a 90KD protein. Occasionally the
transcript by alternate splicing also generates small antigen-t.

~~~~
----T----ß-Ori-àVp1—Vp2—Vp3---
-~~~~

Early
expression of T- antigen leads to replication of the viral DNA using the same
sequences, which are used for early transcription. Early promoters and Late
promoters are reasonably efficient.

By
removing the capsid part of the DNA, it is possible to introduce any desired
gene in its place. By retaining early functional gene this vector can be used
for their episomal expression of a gene product s in a compatible cell line.
By adding signal sequences it is possible to express gene products in secretory
mode.

Recombinant simian virus 40 (rSV40)-derived
vectors are particularly useful for gene delivery to bone marrow progenitor
cells and their differentiated derivatives, certain types of epithelial cells
(e.g., hepatocytes), and central nervous system neurons and microglia. They
integrate rapidly into cellular DNA to provide long-term gene expression in
vitro and in vivo in both resting and dividing cells. Here we describe a
protocol for production and purification of these vectors. These procedures
require only packaging cells (e.g., COS-7) and circular vector genome DNA.
Amplification involves repeated infection of packaging cells with vector
produced by transfection. Cotransfection is not required in any step. Viruses
are purified by centrifugation using discontinuous sucrose or cesium chloride
(CsCl) gradients and resulting vectors are replication-incompetent and contain
no detectable wild-type SV40 revertants. These approaches are simple, give
reproducible results, and may be used to generate vectors that are deleted only
for large T antigen (Tag), or for all SV40-coding sequences capable of carrying
up to 5 kb of foreign DNA. These vectors are best applied to long-term
expression of proteins normally encoded by mammalian cells or by viruses that
infect mammalian cells, or of untranslated RNAs (e.g., RNA interference). The
preparative approaches described facilitate application of these vectors and
allow almost any laboratory to exploit their strengths for diverse gene
delivery applications.

SV40-derived gene therapy Vectors.
http://journals.prous.com/

Vaccinia derived vectors:

In
18th century, Edward Jenner used cowpox for immunizing human beings
against cowpox disease. Since 1850 cow pox and other Vaccinia viruses are used
as live vaccines. Even Fowl pox and Canary poxes are employed in developing
vaccines. In fact canary cowpox recombinant viral vaccines are in phase 1 and
2 trials.

Cow
poxviruses and canary viruses can be used for developing vaccines.

Canary
poxvirus is 250 x 300 nm size. The virus contains 100 or more proteins and it
has lipid envelope with its own specific proteins. The genomic DNA enclosed
is~180 to 200 kbp long, 67% is A/T rich and circularized by single stranded
hairpin loops.

Viral
particles exist in two forms one naked form (NV) and it is found in the
cytoplasm, the second enveloped form that is enveloped with host cellular
membrane with several glycoproteins (EEV).

Nearly
2/3 ds of the genome, central part of it, has replication functions and 1/3rd
of the genome has no replication functions and this region is dispensable. One
of the key enzymes produced by the viral genome is DNA dependent RNA
polymerase, which recognizes unique promoter elements and initiate
transcription.

The
RNA-pol transcribes early class of genes, which are required for DNA
replication. And few others are required for uncoating of the viral particle
for replication.

At
the onset of replication transcription of early genes stop and intermediate
class of genes switch on. Among them three are transcriptional factors, which
are required for late gene transcription. Late gene encodes structural
proteins and early transcription factors and enzymes; all put together are
packed into maturing viral particles. The fourth class of genes is expressed
all the time, for they have early and late promoters upstream of the genes.

Vaccinia
virus;http://en.wikipedia.org/

The Making of "the Unexpected Vector; www.bio.davidson.edu/Courses/Molbio/MolStudents/

As stated above, vaccinia undergoes homologous recombination
during replication in infected cells. When used as an expression vector, this
innate ability to recombine is used to introduce foreign DNA coupled to a
vaccinia promoter, such as tk, into the viral genome (fig. 2). Numerous
variants already exist, including those with indicators such as the lac-Z gene
for blue-white selection (Cann). The steps below outline the construction of
the vaccinia expression vector (fig. 2, by permission of Alan Cann) (Moss).

(1) Your favorite gene (YFG) is flanked with vaccinia DNA sequences,
especially the vaccinia promoters and multicloning sites for cleavage and
ligation. The following are often included:

The
promoters are necessary DNA sequences because the endogenous viral
RNA polymerase binds here to initiate transcription (Unger). The
promoter also determines the direction of translation for the insert, and
more importantly the ability to express proteins (depending on how tightly
regulated the promoter is).

In
addition, DNA sequences, such as the lacO/lacIq repressor system, that act
in conjunction with promoters and also bind repressor molecules can
regulate the induction of transcription (Unger). Hence, by adding or
removing a particular substrate, expression of YFG can be turned on and
off as necessary.

Stabilizing
elements such as transcription terminators can also be incorporated
downstream of the multi-cloning site. These anti-termination elements
signal the RNA polymerase to release the DNA template and stop
transcription, and prevent pausing, pre-mature termination, and over
reading which adversely affect plasmid replication (Unger).

Finally,
small open reading frames, known as ribosome binding sites, upstream of
YFG, can be included to encourage binding and translation of the target
sequence (Unger).

(2) The product (usually a plasmid with an ori and a marker
gene) is then inserted into a cell infected with the whole virus. The whole
virus must be used because it contains the necessary enzymes and factors within
its core.

(3) Recombination during replication leads to insertion of YFG (i.e.
the foreign DNA) into the viral progeny. The usual target of insertion is a
nonessential region, so that virus retains its ability to replicate
independently and the system can be maintained. The estimated incidence of
successful insertion is approximately 0.1% (hey, I didn't say this was easy...).
A major advantage of the Vaccinia vector is that at least 25,000 bp of DNA (a
lot more than most vectors can handle) can be added to the Vaccinia genome
without requiring any deletions.

(4) Controlling when and how much of YFG is expressed is easy
because the poxvirus promoter sequences control the rate and time of
expression, and you can regulate which promoters are in the system. The highest
yields of protein are generally generated with the late promoters.

(5) Virus plaques can finally be screened by DNA hybridization or for
expression of your favorite protein.

With the rapid discovery of new genes, especially from the Human
Genome Project, comes the daunting task of understanding how the products of
these genes are synthesized, regulated, and used within cells. Vaccinia virus,
as a vector for expression systems, is a powerful addition to the range of
molecular methods available for such purposes. The use of Vaccinia allows
temporal, as well as quantitative regulation of protein expression. More
importantly, Vaccinia is large enough to accommodate several gene inserts while
preserving the entire length of its DNA. Finally, as an infectious agent, it
can target specific cells for insertion, and may thus be employed in gene and
cancer therapy. Led by Vaccinia, the Poxviridae may no longer be
considered the scourge of the world, but rather powerful tools for advancing
research and therapeutic avenues.

Various
transfer vectors have been created using different kinds of promoters such as
TK, 7.5K, 11K, CPK. T7-10 etc. Among them late gene promoters are very strong
and efficient promoters, ex. 11K.

By
recombinant methods T7 promoter has been introduced into the Vaccinia genome
and it is the most expressive promoter. However to express it, one has to
introduce T7 RNA pol gene in expression mode. Any foreign gene can be
constructed under T7 promoter. In fact any of the foreign genes can be
inserted into any of the 55 genes, but widely used one is Thymidine Kinase gene
for in vitro manipulation.

First
construct a plasmid with required composition of genes and a segment of DNA for
homologous recombination with wild type virion DNA. The TK-L and TK-R are the
left and right end borders of Thymidine kinase gene from wild type

Infect
cells with the plasmid construct and wild type viral DNA and allow
recombination, which can be screened for the recombinants easily. When grown
in the presence of deoxy 5’ Bromo uracil, the wild type thymidine kinase
undergoes mutation and it does not function, but recombinant has only the
borders and not the entire TK gene, so the recombinants survive and wild type
die. Glycover can be used to screen for the absence of Thymidine kinase.

Another
way to identification, whether or not recombination has taken place or not, is
add b-Galactoside for the b-Galactosidase as the marker gene is expressed in the
recombinant, and the color generated indicated the construct is working as the
recombinant.

Cell
lines used in these protocols are Monkey kidney cells called CV-1 and Rabbit
kidney cell called RK-13.

The
virus has a wide host range. Such antigen expressing recombinant viral DNA can
be used as live vaccines.

Replication
of the viral DNA takes place in the cytoplasm and not in the nucleus. The
viral derived vectors have been used to develop live recombinant viruses.
These viruses are also used for developing vaccines against Rabies G protein,
Hepatitis B surface antigen, Influenza NP and HA proteins, VSV-N and G
proteins, HSV glycoproteins-type-1. The same were used in clinical trials and
found that the animals immunized show immunity against specific infection.

Vaccinia
has an advantage in cloning a large sized DNA containing different structural
regions of different infectious agents, so as to express as single proteins so
as to obtain immunity against multiple infections!

Adeno Viral derived vectors:

Adenovirus,
though an animal virus, now found to infect human beings. It is now a reality
that quite a number of animal viruses by mutation are infecting human beings.
A good example is Corona virus, the DNA has undergone certain deletion and
changes in nucleotide sequence, and as a consequence this virus has become a
potential killer.

The
virus has wide range of hosts and causes oesophyrengeal infections, not very
serious. The virus is icosahedra in shape, 130nm, and nonenveloped. The DNA
is doubling stranded with sticky ends and 36 to 38 kbp long; and surprisingly
linear, but the binding of 50 KD proteins protects the ends at 5’ region at
either end. The genome has 133bp long terminal repeats. It has a total of 100
map units.

Early
genes expressed are E1, E2, E3 and E4. Early gene-1 is a transcription factor,
which activates several host cell gene expression. They also activate viral
gene expression and help the viral DNA replicative functions. The late genes
code for capsid proteins. During lytic phase the viral DNA is replicates to
several thousand copies and using late gene products they produce ADV particles
and lyse the cells.

Adeno
viral life cycle; infection

The
left end of the genome, nearly 12% has replicative functions. This can be
cloned into cell lines as integrated part. This segment of DNA in human kidney
helper cell lines provides functions for defective recombinant virus.

Early
promoters used are E1a and E1b. An early gene can be replaced with a foreign
gene of interest can be cloned under the promoter of E1a. Transfection of such
DNA into helper cell lines like 293 produce packed ADV particles with
recombinant DNA. Such viruses can produced in large amounts, scaling up of the
production of recombinant viruses is must for application. Such viruses can be
used as nasal sprays in combating certain diseases. Example Cystic fibrosis is
due to the defect in producing a channel protein for the transport of chloride
ions. Recombinant viruses containing cystic fibrosis gene has been cloned and
such ADV’s are used as nasal sprays and found curative effects. The problem is
human body develops immunity against ADV’s, and then the problem remains the
same.

Adenoviruses
can be made into replication-defective vectors by removing the E1 region
located in the left side of the viral DNA, this early transcript encodes for
proteins that activate the transcription of all other viral proteins. Typically,
an expression cassette encoding the therapeutic gene is inserted into the
deleted E1 region and the resultant recombinant viral vector is propagated in a
cell line that expresses the adenoviral E1 genes (see diagram two).

Second generation

To
further limit the leaky expression of viral proteins and expand the capacity of
adenoviral vectors, further deletions have been made in the adenoviral genome.
Viruses with additional deletions in the E2 or E4 regions have been made by a
number of researchers, creating recombinant vectors that have to be propagated
in special complementing cell lines, expressing the E2 or E4 regions, e.g.
293-C7 (E2), 293-ORF6 (E4) or 911-E4.

Third generation

The
entire adenoviral genome, with exception of the essential cis elements (5’ and
3’ ITRs and packaging signal) can be removed to generate recombinant gutted (or
gutless) adenoviral vectors. . They also have a large capacity for exogenous
DNA, being able to package up to 36 kb of transgene. However, their production
is a tricky process, given that they can only be propagated in the presence of
a helper adenovirus.,

The phenomenon of site-specific recombination can be effectively
employed in order to reduce the contaminating levels of helper virus to less
than 0.1%. In this approach the packaging signal of the helper virus is flanked
by two recombination signals (e.g. loxP sites). The virus is then propagated in
a special cell line that expresses the recombination protein that recognises
these signals (e.g. CRE recombinase). The packaging signal is thereby excised
from the helper virus and its genome can no longer be packaged into the capsid
proteins, thus allowing for efficient packaging and production of the vector
genome only. The gutted adenoviral vectors perhaps represent the best hope for
an effective, large capacity vector based on the adenoviruses, however, until
the efficiency of vector production is significantly improved, it is unlikely
that these vectors will be widely applied in the clinic.

ADV vector

Viral
life cycle

Baculo viral vectors:

Baculoviruses
are a diverse group of insect viruses. Autograpaha californica is a
multinuclear polyhedral virus (ACMNPV), and Bombax mori polyhedron viruses
(BMNPV) are just two such examples.

Genome
size of these viruses is about 128 to 200 kbp (ACMNPV =128 KBP) AND IN CIRCULAR
MODE. Insect cell lines used for the expression of viral genomes are Spodofora
fugiperda (Sf-9 cell lines). On infection viruses multiply within epithelial
cells of gut.

Viral
genes are expressed in temporal fashion, such as early, late and very late.

Early
gene products initiate its DNA replication and also activate the expression of
late genes. This results in the multiplication of the genome as well as
viruses, which in turn are budded off as enveloped viruses (Env), which further
infect new set of cells.

Expression
of very late genes is about 18 to 20 hrs after infection, which code for
polyhydrin proteins. At this stage the host cell nuclear membrane proliferates
and primary viruses are occluded among the polyhydrin matrix proteins (29KD).

In
order to use them for expression foreign genes first clone polyhydrin gene
along with flanking regions into a plasmid for recombination purposes.

Invented
by Dr. Frederick M. Boyce, BacMam is a baculovirus-mediated gene transfer
technique that has gained widespread use because of advantages when compared to
other transfection
methods, (for reviews see, Kost, T.A. et al, In addition, BacMam has
been found to have inherent flexibility over stable cell lines,
which has contributed to its adoption as a standard gene transfer technique
(wikipwdia).

~~~--Col.E1ori—I-V-DNA-I----polyhydrin----I-V-DNA-I---Amp^+--~~~

~~~
= End of a circular plasmid.

V-DNA
= Viral DNA as flanking regions.

Then
delete the polyhydrin gene and in its place put a desired gene under a proper
polyhydrin promoter. Then co-infect insect lines with plasmid DNA and the
viral DNA. In insect cells, homologous recombination results in the
incorporation of the desired gene. The viral DNA replicates to 800-1000 copies
per cell and later generates primary virions, which can further infect.

Mouse
IgG against Pseudomonas aeruginosa lipoprotein was expressed in Baculo viral
recombinants. In this case Transfer vector was constructed in such a way both
light and heavy chain genes are cloned separately under specific polyhydrin
promoters with flanking DNA from the virus. When wild type viral DNA and the
transfer DNA are co infected into insect cell, homologous recombination results
in the integration of both IgG genes under promoters which are expressed in
insect cells. In order to scale up the production one can directly infect
larvae instead if cells.

For
example recombinant Trichoplusia larvae containing human Adenosine deaminase
gene, found the expressed protein level was found to be 3-5% of the cellular
proteins. For continuous production the desired gene has to be to be
constructed under the early gene promoter for they don’t require any viral gene
products for expression. It is also possible to integrate the gene construct
into host cells and express continuously to get more of the recombinant
protein. Advantage of using Baculo viral viruses is one can clone a DNA
fragme4nt of the size of 15-20 kbp long. Direct injecting modified virus can
achieve infection and growth. Proteins expressed in insect are found to have
all characteristic post-translational modification, but in some case
glycosylation is same as that of mammalian system.

This
vector is designed for high level of expression. It consists of strong
polyhydrin vector flanked by homologous sequences for recombination. The MCS
has 18 sites and the other features like M13 origin, Amp^+ and ColE1 Ori.

There
are a large number f RNA viruses, among them retroviruses are important, which
are also called retroviruses. When they infect compatible cells, then virus
replicates and integrates into the host genome as a cDNA but its size is little
longer than the original size of the viral RNA. It is only after activation of
cells, which have the integrated viral genome, produce viral particles.

When
the viral genomic RNA replicates it produces ds cDNA with long terminal repeats
(LTR) at their respective ends, hence the size of this is slightly larger than
the viral genome.

Such
ds viral DNA can be retrieved. The LTR segments and viral packaging sequences
can be used to construct an expression vector. The LTR sequences also contain
promoter elements U3 R U5-Ψ-. In between the LTR segments one can
introduce required genes under specific promoters. And also one can have a
selection marker gene under specific promoter. If the vector has viral
packaging sequences the viral DNA can be packaged into a viral particle and
obtain them in large numbers and the same can be used for infecting a
particular tissue. Upon infection the viral particle release the DNA into
cytoplasm and the circular DNA enters into the Nucleus, where using LTR
sequences the DNA integrates into the host genome. The transfected cells can
be screened with a selection marker gene and the cells can be amplified and the
same can be used to obtain the gene product or the transgenic cells can be used
for genes therapy.

How to develop a live recombinant virus:

In
order to develop a live recombinant virus first one has to transfer viral
capsid genes into specific host cells, where the genes are inserted in
expression mode but regulatable and inducible?. Such cells can be maintained
and expanded; such cells are called helper cell lines.

Such
cells are transfected with above-mentioned viral construct. Then the cells are
stimulated to produce capsid proteins. As the vector DNA has sequences for
packaging, the capsid proteins bind to such sequences and complete packaging
leads to full viral particle production. If such cell line also have specific
envelop protein genes, then the envelope proteins that incorporate into the
viral envelop, which can targeted to specific cell type.

When
the tissue is infected with the recombinant viruses, the released DNA goes into
the nucleus where it gets integrated into the chromosomal DNA using LTR
sequences and rest of the DNA gets degraded.

Application
of this method is very important for one can deliver the viruses into specific
tissues, without causing any adverse effects. Any constructs with LTR
sequences can also used directly transfer the construct into the cells by
direct transfer by any one of the Transfection methods. The transfected DNA
gets integrated into the host genome using LTR sequences. It is greatly
facilitated if the cells are mitotically active. This method has been
employed in gene therapy.

-U3-R-U5-I~~-P---X-I-I-gene—Ttr—I---P—Kan+
--Ttr—U3-R-U5-

U3RU5 = LTR sequences,

~~ = Packaging sequences

II = Introns.

Ttr = transcriptional
terminator.

Design of Retroviral Vectors and Helper Cells for Gene Therapy:

Replication cycle of retroviruses: A
retrovirus binds to a receptor on the cell surface (shown as a crescent),
enters the cell, and reverse transcribes the RNA into double-stranded DNA
(shown as a line flanked by black boxes), viral DNA integrates into the cell
chromosome (shown as zigzag lines) to form a provirus. Cellular machinery
transcribes and processes the RNA (shown as thin lines), and translates the
viral proteins (shown as black ellipses and white circles). Viral RNA and
proteins assemble to form new viruses, which are released from the cell by
budding.

The LinX retroviral expression system created by is an efficient
method for retroviral packaging. It produces amphotropic retrovirus that
infects cells from a broad range of mammalian species. This line is derived from
human embryonic kidney (HEK 293) cell line. The viral gag, pol and env genes-
necessary for particle formation and replication- are stably integrated into
the genome of the LinX packaging cell line. The separate introduction and
integration of the structural genes minimizes the chances of producing
replication-competent virus due to recombination events during cell
proliferation2,3. The retroviral expression vector provides the
viral packaging signal (Ψ), the shRNA to the target gene and a puromycin
resistance marker. Transfection of the pSM2 self-inactivating retroviral vector
into the LinX packaging cell line produces replication-incompetent virus that
can efficiently transfer genes into a variety of mammalian cell types.

Overview of the process of
generating packaged virus particles using shRNA and LinX cells, infection of
target cells and assaying for mRNA or protein expression (readout).

Once the packaging cell line is
transfected with a retroviral expression vector that contains a packaging
signal (1), the viral genomic transcript containing the target gene and
selectable marker are packaged into infectious virus within 48–72 hrs
(2-4). Virus produced in this way can infect target cells and transmit
target genes; however, it cannot replicate within target cells because the
viral structural genes are absent.

The psi-2 Packaging Line of Mann, Mulligan and Baltimore:

The
use of recombinant retroviruses was pioneered by Richard Mulligan and David
Baltimore with the Psi-2 lines and analogous retrovirus packaging systems,
based on NIH 3T3 cells (10, 11). Such helper-defective packaging lines are
capable of producing all the necessary trans proteins--gag, pol, and env-- that
are required for packaging, processing, reverse transcription, and integration
of recombinant genomes. Those RNA molecules that have in cis the Psi packaging
signal are packaged into maturing virions.

Cell
Biolabs Inc. Retroviral Expression and Packaging:

·Platinum (Plat) cell lines provide a
powerful, stable method for producing high levels of retroviral structure
proteins. Plat-E and Plat-A cells stably express the gag, pol,
and env genes, allowing retroviral packaging with a single plasmid
transfection to produce recombinant ecotropic and amphotropic virus, respectively.
Pantropic retrovirus may also be produced by transfecting Plat-GP cells with
the expression vector and a second vector containing VSV-G.

·293RTV cell line is a permanent
cell line established from the parental 293 cell line and selected for high-yield
retroviral production. This cell line stably expresses the SV40 large T
antigen, and will package retrovirus upon transfection with gag, pol, and env
vectors plus an expression vector containing your gene of interest. These
packaging vectors are available individually.

Our
retroviral expression vectors are available with a variety of backbones for
general gene expression purposes or specific transduction of stem cells.
For your convenience, Platinum Retroviral Expression Systems contain both a
Platinum packaging cell line and the vectors required for efficient packaging
of high-titer retrovirus.

GFP (Green Fluorescence protein- (27KD): Produces green light when
exposed to UV or blue light. GFP gene is isolated from Jamaican chick beetle
(Kitty boo called Pyrophorene plagiophthalamus). This jellyfish has two organs
one at abdominal region and the other on its head. It has four genes, which
produce different colors such as green, yellow green, yellow and orange.

There
are a large number of vectors for expression of genes in animal tissues. Many
vectors have been specially created for targeting their products to certain
destination. This is possible if target specific signal sequences are ligated
in frame with a designated gene, the protein produced goes through the
processing and ends in that specific target.